Hypomethylation of long interspersed nuclear element-1 (LINE-1) leads to activation of proto-oncogenes in human colorectal cancer metastasis

Abstract

Objective Hypomethylation of LINE-1 elements has emerged as a distinguishing feature in human cancers. Limited evidence indicates that some LINE-1 elements encode an additional internal antisense promoter, and increased hypomethylation of this region may lead to inadvertent activation of evolutionarily methylation-silenced downstream genes. However, the significance of this fundamental epigenetic mechanism in colorectal cancer (CRC) has not been investigated previously. Design We analysed tissue specimens from 77 CRC patients with matched sets of normal colonic mucosa, primary CRC tissues (PC), and liver metastasis tissues (LM). LINE-1 methylation levels were determined by quantitative bisulfite pyrosequencing. MET, RAB3IP and CHRM3 protein expression was determined by western blotting and IHC. MET proto-oncogene transcription and 5-hydroxymethylcytosine (5-hmc) were evaluated by quantitative real-time-PCR. Results Global LINE-1 methylation levels in LM were significantly lower compared with the matched PC (PC=66.2% vs LM=63.8%; p<0.001). More importantly, we observed that specific LINE-1 sequences residing within the intronic regions of multiple proto-oncogenes, MET (p<0.001), RAB3IP (p=0.05) and CHRM3 (p=0.01), were significantly hypomethylated in LM tissues compared with corresponding matched PC. Furthermore, reduced methylation of specific LINE-1 elements within the MET gene inversely correlated with induction of MET expression in CRC metastases (R=−0.44; p<0.0001). Finally, increased 5-hmc content was associated with LINE-1 hypomethylation. Conclusions Our results provide novel evidence that hypomethylation of specific LINE-1 elements permits inadvertent activation of methylation-silenced MET, RAB3IP and CHRM3 proto-oncogenes in CRC metastasis. Moreover, since 5-hmc content inversely correlated with LINE-1 hypomethylation in neoplastic tissues, our results provide important mechanistic insights into the fundamental processes underlying global DNA hypomethylation in human CRC.

LINE-1 in cancer: multifaceted functions and potential clinical implications

“…L1 expression correlates with tumorigenesis in some cancers and may contribute to the process of transformation in a minority of cases. Moreover, previous studies 32,33,35,37,38 in a small number of cancer types have shown that L1 activity and expression differ among and within cancer types and may fluctuate during cancer evolution, suggesting its potential as a cancer biomarker. Future study is still required to illustrate why L1 activity and expression are deregulated in cancer and how they contribute to tumorigenesis.…”

“…In CRC and HCC, L1 insertion into the intronic region of MET (L1-MET) leads to elevated expression of MET. 38,46 In CRC and associated liver metastasis tissue, the hypomethylation of L1 ASP within L1-MET is correlated with elevated MET expression. 38 In HCC, L1-MET was also identified and its hypomethylation was found to be correlated with elevated c-MET expression.…”

“…38,46 In CRC and associated liver metastasis tissue, the hypomethylation of L1 ASP within L1-MET is correlated with elevated MET expression. 38 In HCC, L1-MET was also identified and its hypomethylation was found to be correlated with elevated c-MET expression. 46 Chromosomal rearrangements and processed pseudogenes.…”

“…Although mounting evidence supports that global hypomethylation involving L1 regions is an important cause of L1 reactivation in cancer, 37,38,59 upstream regulators of this activation remain elusive. Previous studies have suggested some of the upstream regulators, such as oxidative stress, 81,82 interleukin-6, 83 Rad21, 83 and p53 pathway.…”

“…32 In CRC with liver metastasis, L1 methylation level is lower in metastasis versus primary CRC tissue. 38 These two examples indicate that L1 expression can change with tumor progression (also discussed below in "Functions of L1-Encoded Proteins in Cancer"). Target-site analysis revealed that somatic L1 insertions are biased away from transcriptional active regions 35 and toward regions such as intergenic or heterochromatic regions, 37 cancer-specific hypomethylation regions, 35 or genes frequently mutated in cancer, suggesting a possible oncogenic role of L1 insertions given that frequently mutated genes are candidate drivers of tumorigenesis.…”